EP1705250A1 - A METHOD FOR SEPARATING, EXTRACTING AND PURIFYING POLY-BETA-HYDROXYALKANOATES (PHAs) DIRECTLY FROM BACTERIAL FERMENTED BROTH - Google Patents

A METHOD FOR SEPARATING, EXTRACTING AND PURIFYING POLY-BETA-HYDROXYALKANOATES (PHAs) DIRECTLY FROM BACTERIAL FERMENTED BROTH Download PDF

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Publication number
EP1705250A1
EP1705250A1 EP03782063A EP03782063A EP1705250A1 EP 1705250 A1 EP1705250 A1 EP 1705250A1 EP 03782063 A EP03782063 A EP 03782063A EP 03782063 A EP03782063 A EP 03782063A EP 1705250 A1 EP1705250 A1 EP 1705250A1
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Prior art keywords
fermentation liquid
claim1
separating
extracting
liquid
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EP03782063A
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German (de)
French (fr)
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EP1705250B1 (en
EP1705250A4 (en
EP1705250B9 (en
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Xuejun Tianan Biologic Material Co. Ltd. CHEN
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Tianan Biologic Material Co Ltd Ningbo
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Tianan Biologic Material Co Ltd Ningbo
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
    • C08G63/88—Post-polymerisation treatment
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
    • C08G63/02—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds
    • C08G63/06—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds derived from hydroxycarboxylic acids

Definitions

  • This invention relates to post-treatment of biological engineering, particularly to extraction and separation of bacterial fermentation product, or more particularly to extraction and separation of polyhydroxyalkanoates in cells.
  • PHAs Poly- ⁇ -hydroxyalkanoates
  • n and m are 1 ⁇ 4 integer, usually 1, that is 3-hydroxyalkanoates (3-HAS)
  • R 1 and R 2 are straight chain or branched chain C 1-12 alkyl which are substituted or non-substituted
  • X and Y are not 0 simultaneously, and determine the content of the component in copolymer.
  • the average molecular weight of PHAs is generally 1-4 million Da.
  • PHAs The physical property of PHAs is similar to that of polypropylene. As its biodegradability, biocompatibility, piezoelectricity and optical activity are characteristics not possessed by common petrochemical resins, it has wide application prospect in industry, agriculture, medicine, sanitation, food, electronics, etc.
  • the cost of PHAs includes mainly material cost and separation purification cost.
  • the material cost depends on production efficiency of bacteria species and fermentation technology, whilst the separation purification cost depends largely on technology.
  • the current extraction technology includes separation of cells from fermentation liquid with high speed centrifuge and purification of PHAs in separated wet bacterial body with organic solvent extraction, chemical reagent or surfactant + enzyme. These methods have the defect of high cost or serious pollution, and are difficult to be industrialized.
  • the purpose of this invention is to provide an extraction method for PHAs, which can reduce effectively separation and purification cost, reduces pollution, and is suitable for industrialized production.
  • This invention provides a method for directly separating and purifying polyhydroxyalkanoates in cells from bacterial fermentation liquid, comprisings:
  • the sequence of adjusting pH and adding surfactant is interchangeable.
  • step 3 aside from adding anionic surfactant, coagulating agent can be added.
  • the physical method used to break cell wall can be ultrasonic breaking, ball milling or high pressure treatment.
  • the pH of the pretreated fermentation liquid is adjusted to 8-13.
  • the alkaline substance used in adjusting pH can be solid or aqueous solution of NaOH, Na 2 CO 3 , NaHCO 3 or ammonia water.
  • the anionic surfactant can be olefinesulfonate (AOS), fatty alcohol sulfate, fatty alcohol polyoxyethylene-ether sulfate (AES), fatty alcoholpolyoxyethylene ether (AEO), alkylphenol-polyoxyethylene ether, etc., its quantity is 0.5-20% (W/V) of fermentation liquid.
  • AOS olefinesulfonate
  • AES fatty alcohol polyoxyethylene-ether sulfate
  • AEO fatty alcoholpolyoxyethylene ether
  • alkylphenol-polyoxyethylene ether alkylphenol-polyoxyethylene ether
  • the coagulating agent is sodium polyacrylate, modified starch, polyamine, etc., its quantity is 0.5-20% (W/V) of the fermentation liquid.
  • reaction temperature under agitation is 10-70°C and the reaction time 5-60min.
  • Centrifuge, filter-press, vacuum suction filtration, etc. can be used for separating and extracting precipitate from the reaction liquid.
  • the invention is applicable to separation and purification of fermentation liquid of bacteria and its aberrance and gene engineering bacteria containing polyhydroxyalkanoates.
  • Applicable bacteria include Alcaligenes, Pseudomonas, Azotobacter, Rhodospirillum, Methylotrophs, Bacillus, etc.
  • the invention has no high requirement for dry weight of cells and content of PHAs in fermentation liquid.
  • the invention has the advantage of simple technology, low cost, high yield and greatly reduced pollution, so large scale industrialized production can be realized.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Polymers & Plastics (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Extraction Or Liquid Replacement (AREA)

Abstract

The invention discloses a method for directly separating and purifying polyhydroxyalkanoates in cells from fermentation liquid and it relates to the field of posttreatment technology of biological engineering. The method includes following steps: pretreating fermentation liquid with physical method for breaking cell wall; adjusting pH value of pretreated fermentation liquid to alkaline; adding anionic surfactant; reacting under agitation; separating and extracting coagulated precipitate from reaction liquid; washing and drying. The invention has the advantage of simple technology, low cost, high extraction yield and no pollution, and large scale industrialized production can be realized.

Description

    Technical field
  • This invention relates to post-treatment of biological engineering, particularly to extraction and separation of bacterial fermentation product, or more particularly to extraction and separation of polyhydroxyalkanoates in cells.
    • Background of invention
  • Poly- β -hydroxyalkanoates (PHAs) are biological polyesters accumulated in cells by special microorganisms under special growth conditions.
    Figure imgb0001
     In which, n and m are 1~4 integer, usually 1, that is 3-hydroxyalkanoates (3-HAS); R1 and R2 are straight chain or branched chain C1-12 alkyl which are substituted or non-substituted; X and Y are not 0 simultaneously, and determine the content of the component in copolymer. The average molecular weight of PHAs is generally 1-4 million Da.
  • The physical property of PHAs is similar to that of polypropylene. As its biodegradability, biocompatibility, piezoelectricity and optical activity are characteristics not possessed by common petrochemical resins, it has wide application prospect in industry, agriculture, medicine, sanitation, food, electronics, etc.
  • Large scale industrialized production of PHAs has not been realized internationally. The principal reason is the cost is much higher than that of petrochemical resin. The cost of PHAs includes mainly material cost and separation purification cost. The material cost depends on production efficiency of bacteria species and fermentation technology, whilst the separation purification cost depends largely on technology. The current extraction technology includes separation of cells from fermentation liquid with high speed centrifuge and purification of PHAs in separated wet bacterial body with organic solvent extraction, chemical reagent or surfactant + enzyme. These methods have the defect of high cost or serious pollution, and are difficult to be industrialized. One step extraction separation method for extracting polyhydroxyalkanoates directly from fermentation liquid containing cells is disclosed in Chinese patent application CN1328160A , but it must use large quantity of sodium hypochlorite and has the defect of poor operation environment, serious pollution, cost increased by waste water treatment, and product quality affected by shear degradation of PHAs.
  • The purpose of this invention is to provide an extraction method for PHAs, which can reduce effectively separation and purification cost, reduces pollution, and is suitable for industrialized production.
    • Invention
  • This invention provides a method for directly separating and purifying polyhydroxyalkanoates in cells from bacterial fermentation liquid, comprisings:
    1. (1) pretreating fermentation liquid with physical method for breaking cell wall;
    2. (2) adjusting the pH value of the pretreated fermentation liquid so that it is alkaline;
    3. (3) adding anionic surfactant and agitating;
    4. (4) separating and extracting precipitate in reaction liquid;
    5. (5) washing and drying.
  • The sequence of adjusting pH and adding surfactant is interchangeable.
  • In step 3, aside from adding anionic surfactant, coagulating agent can be added.
  • The physical method used to break cell wall can be ultrasonic breaking, ball milling or high pressure treatment.
  • The pH of the pretreated fermentation liquid is adjusted to 8-13. The alkaline substance used in adjusting pH can be solid or aqueous solution of NaOH, Na2CO3, NaHCO3 or ammonia water.
  • The anionic surfactant can be olefinesulfonate (AOS), fatty alcohol sulfate, fatty alcohol polyoxyethylene-ether sulfate (AES), fatty alcoholpolyoxyethylene ether (AEO), alkylphenol-polyoxyethylene ether, etc., its quantity is 0.5-20% (W/V) of fermentation liquid.
  • The coagulating agent is sodium polyacrylate, modified starch, polyamine, etc., its quantity is 0.5-20% (W/V) of the fermentation liquid.
  • After adding the anionic surfactant and the coagulating agent, the reaction temperature under agitation is 10-70°C and the reaction time 5-60min.
  • Centrifuge, filter-press, vacuum suction filtration, etc. can be used for separating and extracting precipitate from the reaction liquid.
  • The invention is applicable to separation and purification of fermentation liquid of bacteria and its aberrance and gene engineering bacteria containing polyhydroxyalkanoates. Applicable bacteria include Alcaligenes, Pseudomonas, Azotobacter, Rhodospirillum, Methylotrophs, Bacillus, etc. The invention has no high requirement for dry weight of cells and content of PHAs in fermentation liquid. The invention has the advantage of simple technology, low cost, high yield and greatly reduced pollution, so large scale industrialized production can be realized.
    • Detailed description of the invention
  • Following examples are used to further describe the invention. These examples should not constitute any limitation to the scope of claims. Any modifications or changes made by the skilled man in the art benefit from the disclosure of this application should be included within the scope of claims stated in this application.
    • Example 1
  • Take 1000ml of fermentation liquid of Alcaligenes entrophus mutant 65-7, in which the dry weight of cells is 142g/l, the content of PHBV is 78.5%; pretreat with ball mill (530r/min, 0.1 mm steel ball) for 40min; adjust pH value to 12 with 30% NaOH solution; add 13g of sodium laurylsulfate; adjust reaction temperature to 32°C; react under agitation for 5min; filter with suction and filter paper; wash precipitate with water till washing becomes neutral; dry at 70°C to constant weight. Purity of the product is 98.2%, the average molecular weight is 5.2x105Da, the yield is 85.2%. the COD and BOD of waste water from suction filtration after treatment with anaerobic and aerobic bacteria is 800 and 30mg/l respectively, in conformity with state discharge standard.
    • Example 2
  • Take 100ml of fermentation liquid of Alcaligenes entrophus, in which the dry weight of cells is 147g/l, the content of PHBV is 75.2%; break cell wall with ultrasonic (1500W) for 20min; adjust pH value to 8 with 30% NaOH solution; add 0.5g of sodium laurylsulfate and 5g of sodium polyacrylate; adjust reaction temperature to 70°C; react under agitation for 30min; filter with suction and filter paper; wash coagulated precipitate with water till washing becomes neutral; dry in oven at 70°C to constant weight. Purity of the product is 93.2%, the average molecular weight is 4.1x105Da, the yield is 80.3%.
    • Example 3
  • Take 50ml of fermentation liquid of Alcaligenes entrophus, in which the dry weight of cells is 102g/l (in which the content of PHB is 60%); pretreat with ball mill (560r/min, 0.1 mm steel ball) for 30min; adjust pH value to 13 with NH3.H2O solution; add 10g of sodium laurylsulfate and 10g of modified starch; adjust reaction temperature to 10°C; react under agitation for 10min; separate with centrifuge (separation factor 600); wash precipitate with water till washing becomes neutral; dry in oven at 70°C to constant weight. Purity of the product is 98.2%, the average molecular weight is 4.4x105Da, the yield is 87%.
    • Example 4
  • Take 500ml of fermentation liquid of Alcaligenes entrophus mutant 65-7, in which the dry weight of cells is 135g/l, the content of PHB is 75.5% and introduce it into a special vessel. Increase pressure to 60MPa, release pressure rapidly after 10min, collect the liquid and repeat the operation twice. Adjust pH value to 10 with 30% NaOH solution; add 9g of sodium lauryl polyoxyethylene ether sodium sulfate; adjust reaction temperature to 38°C; react under agitation for 8min; filter with suction and filter paper; wash precipitate with water till washing becomes neutral; dry at 70°C to constant weight. Purity of the product is96.7%, the average molecular weight is 4.2x105Da, the yield is 81.5%.
    • Example 5
  • Take 100ml of fermentation liquid of Alcaligenes entrophus, in which the dry weight of cells is 154g/l (in which content of PHBV is 80.5%); break cell wall with ultrasonic (2800W, continuous treatment) for 40min; adjust pH value to 11 with 30% NaOH solution; add 10kg of sodium laurylsulfate and 0.5kg of sodium polyacrylate; adjust reaction temperature to 50°C; react under agitation for 60min; filter with filter press; wash precipitate with water till washing becomes neutral; dry in oven at 70°C to constant weight. Purity of the product is 97%, the average molecular weight is 5.3x105Da, the yield is 84%.
    • Example 6
  • Take 100ml of fermentation liquid of Pseudomonas, in which dry weight of cells is 86g/l, content of PHBV is 61.5%; pretreat with ball mill (560r/min, 0.1 mm steel ball) for 50min; adjust pH value to 11 with 30% NaOH solution; add 3g of sodium laurylsulfate; adjust reaction temperature to 24°C; react under agitation for 10min; filter with suction and filter paper; wash precipitate with water till washing becomes neutral; dry at 70°C to constant weight. Purity of the product is 94.2%, the average molecular weight is 3.2x105Da, the yield is 71.2%.

Claims (9)

  1. A method for directly separating and purifying polyhydroxyalkanoates in cells from bacterial fermentation liquid, comprisings:
    (1) pretreating of the fermentation liquid with physical method to break cell wall;
    (2) adjusting pH value of the pretreated fermentation liquid to alkaline;
    (3) adding anionic surfactant and reacting under agitation;
    (4) separating and extracting precipitate from the reaction liquid;
    (5) washing and drying;
    Wherein the sequence of adjusting pH and adding surfactant is interchangeable.
  2. The method according to claim1, wherein coagulating agent can be added in the step (3).
  3. The method according to claim1, wherein physical method can be selected from ultrasonic breaking, ball milling or high pressure treatment.
  4. The method according to claim1, wherein pH of the pretreated fermentation liquid is adjusted to 8-13.
  5. The method according to claim1 or 4, wherein the alkaline substance used to adjust pH is NaOH, Na2CO3, NaHCO3 solid/aqueous solution or ammonia water.
  6. The method according to claim1, wherein the anionic surfactant is olefin sulfonate, fatty alcohol sulfate, fatty alcohol polyoxyethylene ether sulfate, fatty alcohol polyoxyethylene ether or alkylphenol polyoxyethylene ether, its quantity is 0.5-20% (W/V) of the fermentation liquid.
  7. The method according to claim1, wherein the coagulating agent is selected from sodium polyacrylate, modified starch or polyamine, its quantity is 0.5-20% (W/V) of the fermentation liquid.
  8. The method according to claim1, wherein the reaction temperature under agitation is 10-70°C, the time is 5-60min.
  9. The method according to claim1, wherein the method for separating and extracting precipitate from reaction liquid is selected from centrifuge, filter press or vacuum suction filtration.
EP03782063.6A 2003-12-19 2003-12-19 A method for separating, extracting and purifying poly-beta-hydroxyalkanoates (phas) directly from bacterial fermented broth Expired - Lifetime EP1705250B9 (en)

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WO2013016566A1 (en) * 2011-07-26 2013-01-31 Micromidas Inc. Methods of extracting polyhydroxyalkanoates from pha-containing bacterial cells
WO2022090960A1 (en) 2020-10-30 2022-05-05 Biotrend - Inovação E Engenharia Em Biotecnologia, S.A. Process for extraction and purification of polyhydroxyalkanoates
IT202200013483A1 (en) 2022-06-27 2023-12-27 Versalis Spa PROCEDURE FOR THE RECOVERY AND PURIFICATION OF POLYHYDROXYALKANOATES FROM A FERMENTATION BROTH.

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US7968657B2 (en) * 2006-10-21 2011-06-28 Polyone Corporation Thermoplastic polyhydroxyalkanoate compounds
JP2008193940A (en) * 2007-02-13 2008-08-28 Honda Motor Co Ltd Method for purifying polyhydroxybutyrate
US8680192B2 (en) * 2008-07-29 2014-03-25 Polyone Corporation Crystallized thermoplastic polyhydroxyalkanoate compounds
MY155003A (en) * 2009-01-13 2015-08-28 Plainexus Res Lab Sdn Bhd A method for producing biodegradable resins
MY153891A (en) * 2010-03-01 2015-04-15 Univ Putra Malaysia A method for recovering an intracellular polyhydroxyalkanoate (pha)
WO2015133887A1 (en) * 2014-03-06 2015-09-11 Lay Pee Ling Process for the production of biopolymer from waste fish oil or waste palm oil
JP6864585B2 (en) * 2017-08-30 2021-04-28 株式会社カネカ Method for producing polyhydroxy alkanoate
CN109504715A (en) * 2017-09-15 2019-03-22 北京蓝晶微生物科技有限公司 A method of preparing polyhydroxyalkanoate (PHA)
CN111346580B (en) * 2020-04-29 2020-12-11 吉林中粮生化有限公司 Method and system for extracting polyhydroxyalkanoate by combining high temperature and high pressure with ultrasound
CN111500650B (en) * 2020-06-30 2020-10-23 中粮营养健康研究院有限公司 Method for efficiently producing PHA
US20240010789A1 (en) * 2020-11-24 2024-01-11 Kaneka Corporation Poly(3-hydroxyalkanoate) production method
CN112552500B (en) * 2021-01-04 2022-12-20 珠海麦得发生物科技股份有限公司 Method for removing endotoxin in PHAs by fermentation method
CN115058461B (en) * 2022-06-20 2024-05-28 宁波天安生物材料有限公司 Method for directly separating and purifying polyhydroxyalkanoate from fermentation broth
CN115322342B (en) * 2022-08-12 2023-05-26 哈尔滨工业大学 Method for sequentially and synchronously recycling polyhydroxyalkanoate and alginate in activated sludge
CN115786411B (en) * 2023-01-09 2023-06-23 北京微构工场生物技术有限公司 Extraction method of polyhydroxyalkanoate

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013016566A1 (en) * 2011-07-26 2013-01-31 Micromidas Inc. Methods of extracting polyhydroxyalkanoates from pha-containing bacterial cells
WO2022090960A1 (en) 2020-10-30 2022-05-05 Biotrend - Inovação E Engenharia Em Biotecnologia, S.A. Process for extraction and purification of polyhydroxyalkanoates
IT202200013483A1 (en) 2022-06-27 2023-12-27 Versalis Spa PROCEDURE FOR THE RECOVERY AND PURIFICATION OF POLYHYDROXYALKANOATES FROM A FERMENTATION BROTH.
WO2024003698A1 (en) 2022-06-27 2024-01-04 Versalis S.P.A. Process for recovering and purifying polyhydroxyalkanoates from a fermentation broth

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CA2550204C (en) 2012-08-28
CA2550204A1 (en) 2005-06-30
AU2003292860B2 (en) 2010-03-04
EP1705250B1 (en) 2013-06-26
EP1705250A4 (en) 2007-03-21
US7582456B2 (en) 2009-09-01
EP1705250B9 (en) 2013-12-04
WO2005059153A1 (en) 2005-06-30
AU2003292860A1 (en) 2005-07-05
JP2007524345A (en) 2007-08-30
JP4777778B2 (en) 2011-09-21

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